Technical Field
The present disclosure is directed, generally, to the detection of fungal pathogens in a patient sample. More specifically, disclosed herein are methods for detecting and/or identifying a fungal pathogen in a patient sample, involving isolating the sample, optionally extracting DNA from the sample, carrying out a PCR reaction on the sample to generate an amplicon that includes a region of the fungal ribosomal RNA (rRNA) gene, such as an internal transcribed spacer 1 (ITS-1) region and/or a 28S rRNA gene region, and detecting the PCR amplicon. The present disclosure also provides primers and primer sets for specifically detecting a broad range of fungal pathogens in the presence of human ribosomal DNA (rDNA). In certain embodiments of the present disclosure, the PCR amplicon is further characterized by sequencing or by using two-dimensional melt-curve analysis. In yet other embodiments, more than one fungal pathogen is detected in a sample using the methods disclosed herein. The present disclosure also provides methods for identifying alternative primers that are useful for detecting fungal pathogens, and for detecting fungal pathogens in the presence of non-fungal DNA.
Description of the Related Art
Fungal infections remain a major cause of morbidity and mortality in immunocompromised patients, such as those undergoing cancer chemotherapy, solid organ transplants, or hematopoietic cell transplants. The rapid detection and accurate identification of fungal pathogens can be critical for initiating treatment in the earliest stages of infection and for guiding antifungal therapy. Cultivation and histological analysis often have poor diagnostic sensitivity, and histopathological findings frequently do not distinguish among fungal species [McLintock and Jones (2004) Br. J. Haematol. 126:289-97; Reichenberger et al. (1999) Bone Marrow Transplant 24:1195-9]. Moreover, some molecular diagnostic tests such as the galactomannan antigen assay detect only pathogens from the Aspergillus genus, and the beta-glucan antigen assay does not detect fungi in the Zygomycete or Basidiomycete taxa [Kedzierska et al. (2007) Eur. J. Clin. Microbiol. Infect. Dis. 26:755-66; McLintock and Jones (2004) Br. J. Haematol. 126:289-97; Ostrosky-Zeichner et al. (2005) Clin. Infect. Dis. 41:654-9; Yeo and Wong (2002) Clin. Microbiol. Rev. 15:465-84]. Such shortcomings may lead to more empiric antifungal therapy because a fungal infection is not completely excluded with negative results from either of these antigen assays. In addition, the spectrum of fungal infections is likely to change with increasing use of antifungal medications for prophylaxis. The next generation of diagnostic tests must be capable of detecting these emerging pathogens. Finally, pathogenic fungi within the same genus may have different antifungal susceptibility profiles, such as Candida albicans and Candida krusei. 
PCR assays for the detection of fungal pathogens are an appealing approach due to their potential for rapid, sensitive, and accurate diagnosis of fungal infections. Ribosomal RNA genes are particularly attractive targets because they are present in multiple copies per genome, have conserved regions for designing broad-range primers, and more variable regions for identifying fungi. Most studies have focused on 18S rRNA genes [Einsele et al. (1997) J. Clin. Microbiol. 35:1353-60], internal transcribed spacers (ITS1 and ITS2) [Bergman et al. (2007) Eur. J. Clin. Microbiol. Infect. Dis. 26:813-8; Chen et al. (2001) J Clin Microbiol 38:2302-10; Iwen et al. (2002) Med. Mycol. 40:87-109; Turenne et al. (1999) J. Clin. Microbiol. 37:1846-519  and the 5′ end of the 28S rRNA gene (D1-D2 hypervariable region) [(Hinrikson et al. (2005) J. Clin. Microbiol. 43:2092-103; Kurtzman and Robnett (1997) J. Clin. Microbiol. 35:1216-23; Rakeman et al. (2005)J. Clin. Microbiol. 43:3324-33; Sandhu et al. (1995) J. Clin. Microbiol. 33:2913-9; Vollmer et al. (2008) J. Clin. Microbiol. 46:1919-26)] for developing broad-range PCR assays targeting human fungal pathogens.
While certain PCR primers and methods have been developed based on amplification of fungal ITS and D1 -D2 regions within the rRNA operon, there are critical limitations of these primers and the approach. First, these PCR primers have not been designed to prevent the interaction with human DNA. The amplification of human DNA in a patient sample substantially diminishes the utility of such PCR primers thereby compromising the sensitivity and/or specificity of methods for the detection of a fungal pathogen in a human sample. Many of these fungal primers have a high degree of sequence similarity (or are exact matches) with human rRNA genes. Second, there can be intraspecies variability for the ITS regions which could lead to inconclusive species identification in the absence of more complete ITS sequence information in public databases [(Chen et al. (2000) J Clin Microbiol 38:2302-10; O'Donnell et al. (1998) Mycologia 90:465-493; Rakeman et al. (2005) J. Clin. Microbiol. 43:3324-33)]. Third, variability in ITS sequence length could result in inconsistent analytical sensitivity of the fungal PCR assay. For instance, an ITS assay may produce a 200 by amplicon from one fungus, and a 600 by amplicon from a second fungus. The detection assay thresholds for these two fungi are not likely to be the same.
What is critically needed in the art are compositions and methods for achieving the rapid detection and identification of a broad-range of fungal pathogens in patient samples without interference from or interaction with human DNA.